Bottom Line:
The chemical composition, total phenolic content, DPPH radical scavenging activity, Ferric-reducing antioxidant power and tyrosinase inhibition activity were compared between FRP and PSP.Antioxidant and pro-oxidant effect as well as antitumor effect of the extract of FRP and PSP were analyzed by in vitro cell system as well.Moreover, In vitro cell system analysis confirmed that FRP extract exerts comparable activity with PSP, especially, the sub-fraction sch of FRP showed better antioxidant activity at low dosage and stronger per-oxidant activity at high dosage, and both sch of FRP and PSP can dose-dependent induce HepG2 cells apoptosis, which implied tumor therapeutic effect.

ABSTRACTThis study was carried out to evaluate the utilization probability of the fibrous root part (FRP) of Bletilla striata, which was usually discarded and harvesting pseudobulb part (PSP). The chemical composition, total phenolic content, DPPH radical scavenging activity, Ferric-reducing antioxidant power and tyrosinase inhibition activity were compared between FRP and PSP. Antioxidant and pro-oxidant effect as well as antitumor effect of the extract of FRP and PSP were analyzed by in vitro cell system as well. Thin layer chromatography and high performance liquid chromatography analysis indicated that the chemical compositions in the two parts were similar, but the content in FRP was much higher than PSP. Meanwhile, the FRP extracts showed higher phenolic content, stronger DPPH scavenging activity, Ferric-reducing antioxidant capacity and tyrosinase inhibition activity. Sub-fraction analysis revealed that the distribution characteristic of phenolic components and other active constituents in FRP and PSP were consistent, and mainly deposited in chloroform and acetoacetate fractions. Especially, the chloroform sub-fraction (sch) of FRP showed extraordinary DPPH scavenging activity and tyrosinase inhibition activity, with IC50 0.848 mg/L and 4.3 mg/L, respectively. Besides, tyrosinase inhibition activity was even stronger than the positive compound arbutin (31.8 mg/L). Moreover, In vitro cell system analysis confirmed that FRP extract exerts comparable activity with PSP, especially, the sub-fraction sch of FRP showed better antioxidant activity at low dosage and stronger per-oxidant activity at high dosage, and both sch of FRP and PSP can dose-dependent induce HepG2 cells apoptosis, which implied tumor therapeutic effect. Considering that an additional 0.3 kg FRP would be obtained when producing 1.0 kg PSP, our work demonstrated that FRP is very potential to be used together with PSP.

pone-0058004-g007: Effect of sub-fractions on intracellular ROS levels in HepG2 cell line.HepG2 cells were stimulated for 1.5 h, intracellular ROS levels were assessed as described in the ‘Materials and Methods’ section. (A) Dose-dependent effect of H2O2 on intracellular ROS levels; (B,C and D) Intracellular ROS levels in cultured HepG2 in the absence (CTRL) or presence of the indicated concentration of (B) spe, (C) sch and (D) sac sub-fractions of both FRP and PSP. (A–D) * Significantly different from the control, P<0.05; n = 3.

Mentions:
To validate the ROS assay, the variations of intracellular ROS levels in response to increasing doses of H2O2 was detected in HepG2 cell line model. Data in Fig. 7A showed that the fluorescence signal increased in response to H2O2 (a well-known pro-oxidants) in a dose-dependent manner, and then the effect of the five sub-fractions of both FRP and PSP on the intracellular ROS levels were determined; results were expressed as a percentage of controls. Treatment of HepG2 cells with sub-fractions spe, sch and sac of both FRP and PSP exerted a significant antioxidant effect at low dosage (Fig. 7B, 7C and 7D), which confirmed the protective effect of Bletilla striata. Corresponding to the chemical assay system, the sch sub-fractions manifested prominent antioxidant effect below 10 µg/ml. However, the exposure of cell cultures to higher concentrations of spe, sch and sac all increased intracellular ROS levels in dose-dependent manner. These results were distinct to that of previous cell free system. As many publications reported, the antioxidant effect of those sub-fractions (spe, sch and sac) at low dosage was lost and a marked pro-oxidant effect was evident at high dosage [20], [35]; which may imply that compounds impact intracellular ROS levels mainly through compounds-cellular receptor or compounds-cellular signal transductions way, rather than compounds-ROS direct interaction way. Additionally, the sub-fractions sbu and swa for both FRP and PSP showed no significant antioxidant activity.

pone-0058004-g007: Effect of sub-fractions on intracellular ROS levels in HepG2 cell line.HepG2 cells were stimulated for 1.5 h, intracellular ROS levels were assessed as described in the ‘Materials and Methods’ section. (A) Dose-dependent effect of H2O2 on intracellular ROS levels; (B,C and D) Intracellular ROS levels in cultured HepG2 in the absence (CTRL) or presence of the indicated concentration of (B) spe, (C) sch and (D) sac sub-fractions of both FRP and PSP. (A–D) * Significantly different from the control, P<0.05; n = 3.

Mentions:
To validate the ROS assay, the variations of intracellular ROS levels in response to increasing doses of H2O2 was detected in HepG2 cell line model. Data in Fig. 7A showed that the fluorescence signal increased in response to H2O2 (a well-known pro-oxidants) in a dose-dependent manner, and then the effect of the five sub-fractions of both FRP and PSP on the intracellular ROS levels were determined; results were expressed as a percentage of controls. Treatment of HepG2 cells with sub-fractions spe, sch and sac of both FRP and PSP exerted a significant antioxidant effect at low dosage (Fig. 7B, 7C and 7D), which confirmed the protective effect of Bletilla striata. Corresponding to the chemical assay system, the sch sub-fractions manifested prominent antioxidant effect below 10 µg/ml. However, the exposure of cell cultures to higher concentrations of spe, sch and sac all increased intracellular ROS levels in dose-dependent manner. These results were distinct to that of previous cell free system. As many publications reported, the antioxidant effect of those sub-fractions (spe, sch and sac) at low dosage was lost and a marked pro-oxidant effect was evident at high dosage [20], [35]; which may imply that compounds impact intracellular ROS levels mainly through compounds-cellular receptor or compounds-cellular signal transductions way, rather than compounds-ROS direct interaction way. Additionally, the sub-fractions sbu and swa for both FRP and PSP showed no significant antioxidant activity.

Bottom Line:
The chemical composition, total phenolic content, DPPH radical scavenging activity, Ferric-reducing antioxidant power and tyrosinase inhibition activity were compared between FRP and PSP.Antioxidant and pro-oxidant effect as well as antitumor effect of the extract of FRP and PSP were analyzed by in vitro cell system as well.Moreover, In vitro cell system analysis confirmed that FRP extract exerts comparable activity with PSP, especially, the sub-fraction sch of FRP showed better antioxidant activity at low dosage and stronger per-oxidant activity at high dosage, and both sch of FRP and PSP can dose-dependent induce HepG2 cells apoptosis, which implied tumor therapeutic effect.

ABSTRACTThis study was carried out to evaluate the utilization probability of the fibrous root part (FRP) of Bletilla striata, which was usually discarded and harvesting pseudobulb part (PSP). The chemical composition, total phenolic content, DPPH radical scavenging activity, Ferric-reducing antioxidant power and tyrosinase inhibition activity were compared between FRP and PSP. Antioxidant and pro-oxidant effect as well as antitumor effect of the extract of FRP and PSP were analyzed by in vitro cell system as well. Thin layer chromatography and high performance liquid chromatography analysis indicated that the chemical compositions in the two parts were similar, but the content in FRP was much higher than PSP. Meanwhile, the FRP extracts showed higher phenolic content, stronger DPPH scavenging activity, Ferric-reducing antioxidant capacity and tyrosinase inhibition activity. Sub-fraction analysis revealed that the distribution characteristic of phenolic components and other active constituents in FRP and PSP were consistent, and mainly deposited in chloroform and acetoacetate fractions. Especially, the chloroform sub-fraction (sch) of FRP showed extraordinary DPPH scavenging activity and tyrosinase inhibition activity, with IC50 0.848 mg/L and 4.3 mg/L, respectively. Besides, tyrosinase inhibition activity was even stronger than the positive compound arbutin (31.8 mg/L). Moreover, In vitro cell system analysis confirmed that FRP extract exerts comparable activity with PSP, especially, the sub-fraction sch of FRP showed better antioxidant activity at low dosage and stronger per-oxidant activity at high dosage, and both sch of FRP and PSP can dose-dependent induce HepG2 cells apoptosis, which implied tumor therapeutic effect. Considering that an additional 0.3 kg FRP would be obtained when producing 1.0 kg PSP, our work demonstrated that FRP is very potential to be used together with PSP.